Five major approaches have been taken to define non-classical multidrug resistance in cancer. In the first, we isolated KB cells (a subclone of HeLa) resistant to increasing levels of cisplatin and demonstrated multidrug resistance to arsenite and cadmium, to methotrexate, and to nucleoside analogs. This cross-resistance pattern is due to reduced uptake of each of these agents because their receptors have been relocalized from the cell surface into the cytoplasm of the cell. This relocalization of surface transporters appears to be due to altered recycling of these transporters due to alterations in the cytoskeleton that affect endocytic recycling compartments in cisplatin-resistant cells. Overexpression of the negative transcription regulator GCF2 occurs in cisplatin-resistant lines, which reduces expression of rhoA, causing disruption of the cytoskeleton as a proximate cause of this recycling defect. One additional consequence of reduced cell surface transporters is a reduction in glucose uptake and altered mitochondrial metabolism mediated by SIRT1. To determine additional molecular defects that lead to cisplatin resistance, we created a cDNA library from resistant cells and transfected it into sensitive cells to determine which genes confer multidrug resistance, including resistance to cisplatin. Several cDNAs, including those encoding metallotheinein, heat shock proteins, ribosomal proteins, a selenoprotein, and the trans-membrane protein MBC3205 were identified from this selection and their role in cisplatin resistance has been demonstrated. Expression of MBC3205, a membrane protein expressed in normal secretory cells, in combination with the small GTPase Rab8, confers cisplatin resistance. There are also changes in specific micro RNAs (miRNAs) consistently seen in cisplatin-resistant KB cells, and their contribution to the drug resistance is being explored. A second approach was to evaluate the unique features of melanoma cells that contribute to multidrug-resistance. One obvious feature of melanoma cells is the melanosome, a lysosome-derived organelle in which pigment formation takes place. We have shown that cisplatin is sequestered in this organelle, independent of extent of melanin formation, and extruded with melanosomes into the medium, reducing nuclear accumulation of this anti-cancer drug. Evidence indicating that type II and III melanosomes, and not type I or type IV melanosomes, contribute more to drug resistance suggests that the melanosomal maturation pathway could be a target for sensitizing melanomas to chemotherapy. Studies are underway to determine whether ABCB5, a transporter homologous to ABCB1, expressed at high levels in pigmented cells such as melanocytes and melanomas, contributes to the melanosomal sequestration seen in melanomas. Full-length ABCB5 has been expressed in KB cells, where it confers a broad multidrug resistance phenotype. A third approach is to determine the molecular basis of multidrug resistance in cancer stem cells. As part of an NIH Breast Cancer Consortium, supported in part by breast cancer stamp funds, we have begun to isolate breast cancer stem cells and normal breast epithelial stem cells from surgical samples. CD133 positive cells from these cell populations can be propagated in tissue culture using approaches previously developed for growing human ES cells. These cells have other characteristics of stem cells, such as growth as spheroids and expression ABCG2. Using an in vitro assay system for growth and migration of human breast cancer cells in collagen explants (in collaboration with Josh Zimmerberg, NICHD), we plan to evaluate the biological properties of these putative cancer stem cells. Our goal is to evaluate the expression of multidrug-resistance genes, including both classical ABC efflux transporters and uptake transporters, as well as non-classical mechanisms of multidrug resistance, in cancer stem cells derived from these surgical speciments. The ultimate level of drug accumulation in a cell depends on both the rate of drug uptake as well as the rate of efflux. We have begun to explore the role of known solute carrier (SLC and SLCO) transporters as contributors to patterns of drug sensitivity and resistance in cancer cells. Our initial approach has been to correlate expression of SLC and SLCO transporters with degree of drug sensitivity in the NCI-60 cell lines. Several hits were obtained, indicating that expression of uptake transporters is associated with drug sensitivity. Transfection of SLC22A4 into KB cells increases the uptake of cisplatin, doxorubicin and mitoxantrone and concomitantly confers increased sensitivity to these drugs. In another approach, we have developed a Taqman Low Density Array (TLDA) microfluidic chip to detect mRNA expression of 380 different putative drug resistance genes and demonstrated that it is a sensitive, accurate, reproducible, and robust way to measure mRNA levels in tumor samples. Previous work from our laboratory indicates that mRNA measurements of levels of drug-resistance genes are, to a first approximation, predictive of functional expression of drug-resistance mechanisms. This drug-resistance chip is being applied to analysis of human cancers that show either response or lack of response to specific chemotherapy. We have initiated our studies on ovarian cancer, where cancers frequently respond to chemotherapy and then become resistant, on melanoma, on head and neck cancers, on hepatomas, on glioblastomas and on colon cancer. One early result from this analysis is that existing cancer cell lines do not mimic the expression patterns of actual human cancers for the 380 putative drug resistance genes chosen for the TLDA analysis and the simple expedient of growing cells in 3D culture does not correct this problem. This suggests the need for better &lt;i&gt;in vitro&lt;/i&gt; cancer cell models to study multidrug resistance.